COMBINATION OF 25-HYDROXYVITAMIN D AND ANTI-INFLAMMITORIES FOR SUSTAINED PLASMA 17- ß ESTRADIOL LEVELS

ABSTRACT

This invention relates to the use of the combination of 25-hydroxyvitamin D3 (“25-OH D3”) and antioxidants (ascorbic acid vitamin E and canthaxanthin) to make a premix or feed for use in maintain, increasing, or ameliorating the decrease of 17 β-estradiol levels in poultry which are observed when poultry is fed ad libitum, thereby extending the poultry&#39;s egg laying life.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to the combination of 25-hydroxyvitamin D (“25-OH D3” and/or “25-OH D2”) and antioxidants/anti-inflammatories (ascorbic acid, Vitamin E and canthaxanthin) for use in poultry feed for sustaining 17-β-estradiol levels of female birds, thereby extending their productive lifetime as egg layers. It also relates to feed and feed premixes containing the combination of 25-hydroxyvitamin D and the antioxidants/anti-inflammatories for this use.

BACKGROUND OF THE INVENTION

Reproductive efficiency and the incidence of pathogenic disorders are directly influenced by the extent of ovary development and nutrition in poultry. For example, the finely tuned reproductive system of the broiler breeder female requires ovary, oviduct, brain, liver and skeletal system to communicate among themselves under the influence of lighting in the production of hatching eggs. The hypothalamus, located within the brain, is directly stimulated by light energy at photostimulation. The mature (reproductively functional) hypothalamus then secretes luteinizing hormone releasing hormone (LHRH), which acts on anterior pituitary to stimulate the production of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Both LH and FSH target the ovary and stimulate ovarian hormone production and early follicle development, respectively.

The ovary of an immature pullet has only small undifferentiated follicles invisible to the naked eye. These follicles, upon sexual maturity, increase in size to form an array of small follicles as well as a hierarchy of large follicles varying in size. The small follicles produce estrogen after hypothalamic maturation. The large yellow follicles are usually defined as being greater than 1 cm in diameter, and their numbers are directly influenced by lighting program, plane of nutrition and breeder age. Progesterone released from the largest follicle triggers the ovulation process. Mature follicles are capable of producing progesterone only a few hours prior to ovulation. The liver is a key organ in egg production as it is the site of lipogenesis, leading to formation of fatty acids and subsequently triglycerides that contribute to egg yolk formation, which is in turn driven by estrogen. The skeletal system is intimately associated with egg production for its role in mobilizing, storing and releasing calcium under the influence of estrogen.

Hyperphagia and therefore adiposity occurred in broiler breeder females have increased in parallel with the perpetual genetic selection for rapid growth in broiler chickens. Overfeeding-induced ovarian dysfunction and reproductive inefficiency as well as metabolic disorders like ascites, sudden death syndrome and fatty liver in broiler breeder females is a consequence of lipotoxicity development as result of fuel overloading the biological system. Therefore, the challenge of managing modern female broiler breeder strains is the inability of these hens to adequately self-regulate feed intake during growth and development to achieve an optimal body weight and composition to support efficient egg and chick production. As such, broiler breeder females are subject to a high degree of restricted feeding regimen. The primary mechanism in which feed restriction benefits reproductive efficiency in broiler breeder females is the control of follicle development or rather limiting the formation of excessive numbers of ovarian yellow follicles arranged in multiple hierarchies.

Broiler breeder females overfed during reproductive development not only produce excess large yellow ovarian follicles but also generate a greater number of atretic yellow follicles and commonly display erratic oviposition and defective egg syndrome (EODES) that include several reproductive problems such as follicular atresia, the production of soft-shelled or membranous eggs, double-yolked eggs, egg yolk peritonitis (presence of egg yolk in the abdominal cavity), multiple egg days and oviposition not occurring in sequence, resulting in increased production of unsettable eggs.

Controlled studies reported that voluntary feeding (i.e., broiler breeder hens to satiation) resulted in poor egg production, high rate of mortality and abnormal ovarian structure (mainly overt hierarchical follicle atresia. Lipotoxicity leading to impaired ovarian dysfunctions, including follicle atresia, ovarian regression, and a decline of circulating estradiol levels in feed-satiated hens, was also observed.

Despite restricted feeding regimen strictly implemented in commercial broiler breeder flocks, it is still very easy to overfeed breeder hens due to their intrinsic hyperphagia. Furthermore, breeder farm managers are confronted as to when and how to feed before and during the start of egg production as well as towards, during and after peak production. The basic fundamental question to ask what and how management and nutritional tools breeder farm managers can apply and implement to ameliorate the adverse and deleterious effects of reproductive efficiency associated with obesity in overweight hens.

Hy⋅D® (registered trademark for 25-OH-D3; available from DSM Nutritional Products, Switzerland has been used to promote bone health in poultry.

The combination of 25-OH D3 and canthaxanthin has also been used in poultry. WO2010/057811 (DSM IP ASSETS, BV) describes this combination for use in improving hatchability, fertility, and lower embryo mortality in poultry. The combination is commercially available under the trademark MAXICHICK. There is no mention in the patent publication of the inclusion of ascorbic acid and high vitamin E levels, nor the uses to increase, maintain, or ameliorate the decrease in 17-β-estradiol levels in animals fed ad libitum.

Vitamin C (ascorbic acid) is often not included as a supplement in poultry diets, as the chicken can under normal rearing conditions can produce sufficient Vitamin C. However, it has been used in some specific conditions, such as in heat stress situations.

Vitamin E is generally added to poultry feed. Recommended doses for poultry species tends to range from about 50-100 IU/kg feed, depending on the age of the animal.

WO14/191153 (DSM IP ASSETS B.V) teaches the combination of canthaxanthin and at least one of Vitamin C, Vitamin E, selenium, and optionally at least one of thymol, eugenol, vanillin and gamma-terpinene can improve immune statues, bone health, skeletal development and growth and feed conversion, particularly when flocks are subject to stress associated with vaccination.

WO14/202433 (DSM IP ASSETS B.V) teaches the combination of canthaxanthin and 25-OH D3 to improve internal egg quality, i.e. enhancing the strength of vitelline membrane that envelops the yolk. There is no teaching to add ascorbic acid to the combination, nor for its use in ameliorating the adverse effects of hyperphagia on 17-β estradiol levels.

Thus there is a need to control, maintain, increase, or lessen a decrease of 17-β estradiol levels in poultry.

DETAILED DESCRIPTION OF THE INVENTION

It has been found, accordance with this invention that the combination of 25-hydroxyvitamin D (25-OH D3 and/or 25-OH D2) and antioxidants/anti-inflammatories ameliorates the decrease of 17-β estradiol levels observed when poultry is fed ad libitum or experiences hyperphagia-related obesity. It has also been found in accordance with this invention, that the bio-actives traditionally included in the feed as antioxidants also possess an anti-inflammatory activity which mitigates the drop in 17-β estradiol levels. Thus, one aspect of this invention is the use of 25-OHD and antioxidants/anti-inflammatories to ameliorate a decrease of 17-β estradiol levels of poultry experiencing hyperphagia-related obesity, and thereby extend their productive life as egg layers.

Another aspect of this invention is a method of extending the productive life of poultry which are kept as egg layers comprising adding a combination of 25-OH D and antioxidants/anti-inflammatories to its basal diet.

As 25-OH D2 and 25-OH D3 may act in a similar fashion after administration, it is envisioned that either may be used separately in combination with antioxidants/anti-inflammatories, or a mixture of both 25-OH D3 and 25-OH D2 may be used in combination with antioxidants/anti-inflammatories. If used together, the ratio of 25-OH D3:25-OH D3 is not a critical part of the invention.

The antioxidants/anti-inflammatories of this invention comprise the combination of ascorbic acid, vitamin E and canthaxanthin. Thus one aspect of this invention is the combination of 25-OH D3, canthaxanthin, vitamin E and ascorbic acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels. Another embodiment is the combination of 25-OHD2, canthaxanthin, Vitamin E and ascorbic acid acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels. Another embodiment is the combination of 25-OHD3, 25-OHD2, canthaxanthin, Vitamin E and ascorbic acid acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels.

Another aspect of this invention is the combination of 25-OH D, canthaxanthin, vitamin E and ascorbic acid, which optionally further comprises at least one further bio-active ingredient selected from the group consisting of:

-   -   Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper,         Manganese, and Selenium acid for the use of increasing,         maintaining, or ameliorating 17-β estradiol levels. Preferably         the 25-OH D is 25-OH D3. Preferably at least Vitamin D is a         further bio-active ingredient. Sometimes the further bio-active         ingredients include at least Vitamin D and selenium. In some         cases, all the further bio-active ingredients are added.

A further aspect is the combination of 25-OH D, canthaxanthin, vitamin E and ascorbic acid which optionally further comprises at least one further bio-active ingredient selected from the group consisting of Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, Selenium, and combinations thereof acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels. Preferably the 25-OH D is 25-OH D3. Sometimes, the further bio-active ingredient includes biotin. Sometimes the further bio-active ingredient includes Vitamin D and biotin. Sometimes the further bio-active ingredient includes all the aforementioned optionally bio-active ingredients.

Another aspect of this invention is a poultry feed acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels comprising the combination of 25-OH D2 or 25-OH D3 or mixtures thereof, ascorbic acid, Vitamin E and canthaxanthin.

Yet another embodiment is poultry feed acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels comprising the combination of 25-OH D, canthaxanthin, vitamin E and ascorbic acid, which optionally further comprises at least one further bio-active ingredient selected from the group consisting of: Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, Selenium and combinations thereof. Preferably the 25-OH D is 25-OH D3. Sometimes the further bio-active ingredients include at least Vitamin D and Selenium. In some cases, all the further bio-active ingredients are added.

Another embodiment is poultry feed acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels. comprising the combination of 25-OH D, canthaxanthin, vitamin E and ascorbic acid, which optionally further comprises at least one further bio-active ingredient selected from the group consisting of: Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, Selenium and combinations thereof. Sometimes, the further bio-active ingredient includes biotin. Preferably the 25-OH D is 25-OH D3. Sometimes the further bio-active ingredient includes Vitamin D and biotin. Sometimes the further bio-active ingredient includes all the aforementioned optionally bio-active ingredients.

Another aspect of this invention are premixes for poultry feed acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels which comprise the combination of 25-OH D, vitamin E, ascorbic acid and canthaxanthin. Preferably, the 25-OH D is 25-OH D3. In some embodiments the feed and premix acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels also comprises at least one further bio-active ingredient selected from the group consisting of:

-   -   Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper,         Manganese, Selenium and combinations thereof. Sometimes the         further bio-active ingredients include at least Vitamin D and         Selenium. In some cases, all the further bio-active ingredients         are added.

Another aspect of this invention are premixes for poultry feed acid for the use of increasing, maintaining, or ameliorating 17-β estradiol levels which comprise at least one further bio-active ingredient selected from the group consisting of: Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, Selenium and combinations thereof. Sometimes, the further bio-active ingredient includes biotin. Sometimes the further bio-active ingredient includes Vitamin D and biotin. Sometimes the further bio-active ingredient includes all the aforementioned optionally bio-active ingredients.

When using the poultry feed of this invention, the animals may be fed ad libitum and the ill effects of obesity, including decreasing 17-β estradiol levels will be experienced to a lesser degree or not at all. As lowering 17-β estradiol levels results in a shortening of the poultry's productive egg laying life span, maintaining the 17-β estradiol levels will result in prolonging the poultry's usefulness.

As used throughout this specification and claims, the following definitions apply:

“25-OH D” refers to any form of 25-hydroxyvitamin D (i.e. either 25-OH D2 or 25-OH D3, or mixes thereof). 25-OH D3 specifically refers to 25-hydroxyvitamin D3; 25-OH D2 specifically refers to 25-hydroxyvitamin D2.

“Poultry” means any domesticated fowl, including chickens (including broiler, layers and breeding hens), ducks, geese, turkeys, quail, and ostriches.

“Hyperphagia” is excessive eating; the animal does not voluntarily limit its feeding.

“Ascorbic Acid” and “Vitamin C” are used interchangeably throughout the specification and claims.

“Increasing, maintaining, or ameliorating the decline of 17-β estradiol levels” means that

-   -   the 17-β estradiol level of poultry fed the combination of this         invention ad libitum is higher than that which would be observed         in the same type of poultry fed the same base diet (i.e. the         same diet except for the combination of this invention) ad         libitum.

“Basal diet” means that the feed used supplies the poultry with sufficient vitamins and minerals so that the poultry are vitamin and mineral replete.

It has been found in accordance with this invention, that the use of feed comprising 25-OH D3, vitamin E, canthaxanthin and ascorbic acid can increase, maintain, or ameliorate the decline of 17-β estradiol levels.

Increasing, maintaining or ameliorating the decline of 17-β estradiol levels is important for breeding poultry in that it contributes to the longevity of the period during which the animal is productive.

Doses

25-OH D3: The amount of 25-OH D3 can range from 15-200 μg/kg feed. Preferably, the amount of 25-OH D3 is from 35-150 μg/kg feed. For feed with a low dose of the combination of the invention, 35 μg per kg 25-OH D3/feed is preferred; for food with a medium dose of the combination, 69 μg per kg feed is preferred; and for food with a high dose of 150 μg feed is preferred.

Vitamin E: The amount of Vitamin E can range from 40-400 mg/kg feed. Preferably the amount is 80-300 mg/kg feed. For feed with a low dose of the combination of the invention, 80 mg/kg Vitamin E is preferred. For feed with a medium dose, 150 mg Vitamin E is preferred; for feed with a high dose, 300 mg/kg Vitamin E is preferred.

Canthaxanthin: The amount of canthaxanthin can range from 1-15 mg/kg feed. Preferably the amount is 3-12 mg/kg feed. For feed with a low dose of the combination of the invention, 3 mg/kg canthaxanthin is preferred. For feed with a medium dose, 6 mg canthaxanthin is preferred; for feed with a high dose, 12 mg/kg canthaxanthin is preferred.

Ascorbic Acid: The amount of ascorbic acid can range from 40-400 mg/kg feed. Preferably the amount is 100-300 mg feed. For feed with a low dose of the combination of the invention, 100 mg/kg ascorbic acid is preferred. For feed with a medium dose, 150 mg ascorbic acid is preferred; for feed with a high dose, 300 mg/kg ascorbic acid is preferred.

Thus specific preferred feeds of this invention comprise the following dosages (all amounts are per kg/feed): Preferred Feed #1:

-   -   25-OH D3: 15-200 μg,     -   Vitamin E: 40-400 mg,     -   Canthaxanthin: 1-15 mg; and     -   Ascorbic acid: 40-400 mg.

Preferred Feed #2:

-   -   25-OH D3: 35-150 μg,     -   Vitamin E: 80-300 mg,     -   Canthaxanthin: 3-12 mg, and     -   Ascorbic acid: 80-300 mg

Preferred Feed #3: (Low dose feed) This feed is preferred for mildly obese poultry.

-   -   25-OH D3: 35 μg     -   Vitamin E: 80 mg     -   Canthaxanthin: 3 mg     -   Ascorbic acid: 80 mg.

Preferred Feed #4 (medium dose feed) This feed is preferred for moderately to severely obese poultry:

-   -   25-OH D3: 69 μg     -   Vitamin E: 150 mg     -   Canthaxanthin: 6 mg     -   Ascorbic acid: 150 mg.

Preferred Feed #5 (high dose feed) This feed is preferred for severely obese poultry:

-   -   25-OH D3: 150 μg     -   Vitamin E: 300 mg     -   Canthaxanthin: 12 mg     -   Ascorbic acid: 300 mg.

Optional Additional Ingredients

To each of the feeds listed above, at least one of the additional ingredients may be added. Preferably at least one, and more preferably more than one of the following ingredients are added. In other embodiments, all the following ingredients are added:

-   -   Vitamin D3—generally this is present in poultry diets at         approximately 2500 IU per kg feed. In accordance with this         invention, if desired, the amount of Vitamin D is increased to         at least 3000 IU per kg.     -   Vitamin B2: this can be added at 3-25 mg per kg; preferably from         6-20 mg/kg. For low dose feed, 6 mg/kg is preferred. For medium         dose feed, 14 mg/kg is preferred; and for high dose feed 20         mg/kg is preferred.     -   Niacin: this can be added at 25-300 mg per kg feed. Preferably         it ranges from 60-200 mg/kg. For low dose feed, 60 mg/kg is         preferred. For medium dose feed, 120 mg/kg is preferred; and for         high dose feed 200 mg/kg is preferred.     -   Pantothenic acid: this can be added at 10-120 mg per kg feed.         Preferably it ranges from 15-80 mg/kg. For low dose feed, 15         mg/kg is preferred. For medium dose feed, 30 mg/kg is preferred;         and for high dose feed 80 mg/kg is preferred.     -   Folic acid: this can be added at 1-8 mg per kg feed. Preferably         it ranges from 2-6 mg/kg. For low dose feed, 2 mg/kg is         preferred. For medium dose feed, 4 mg/kg is preferred; and for         high dose feed 6 mg/kg is preferred.     -   Biotin: this can be added at 0.05-1.0 mg/kg feed. Preferably it         ranges from 0.2-0.8 mg/kg. For low dose feed, 0.2 mg/kg is         preferred. For medium dose feed, 0.4 mg/kg is preferred; and for         high dose feed 0.8 mg/kg is preferred.     -   Zinc: this can be added at 50-300 mg/kg feed. Preferably it         ranges from 70-250 mg/kg. For low dose feed, 70 mg/kg is         preferred. For medium dose feed, 125 mg/kg is preferred; and for         high dose feed 250 mg/kg is preferred.     -   Copper: this can be added at 5-50 mg/kg feed. Preferably it         ranges from 10-30 mg/kg. For low dose feed, 10 mg/kg is         preferred. For medium dose feed, 20 mg/kg is preferred; and for         high dose feed 30 mg/kg is preferred.     -   Manganese: this can be added at 50-300 mg/feed. Preferably it         ranges from 80-270 mg/kg. For low dose feed, 80 mg/kg is         preferred. For medium dose feed, 150 mg/kg is preferred; and for         high dose feed 270 mg/kg is preferred.     -   Selenium: this can be added at 0.05-0.6 mg/kg feed. Preferably         it ranges from 0.1-0.4 mg/kg. For low dose feed, 0.1 mg/kg is         preferred. For medium dose feed, 0.2 mg/kg is preferred; and for         high dose feed 0.4 mg/kg is preferred.

In one aspect of this invention the combination of 25-OH D3 and the antioxidants/anti-inflammatories are given to poultry which are vitamin replete rather than vitamin deficient. The vitamin replete status is preferably due to the use of a basal feed which supplies at least the minimum amount of vitamins and minerals for the poultry. The combination of this invention is thus preferably used in addition to the basal diet.

Further, it is envisioned that the combinations specified herein may be added to any commercially available poultry food, and thus the total amount of 25-OHD3 and antioxidants/anti-inflammatories present may be equal to the amount originally present in the food plus the addition as specified herein. It is also envisioned that the 25-OHD3 and antioxidants/anti-inflammatories as specified herein are the sole additions to a basal diet which contains at least the minimum required nutrients for poultry nutrition.

While not wishing to be bound by theory, it appears that the increase egg-laying life observed in hens fed the combination of this invention could possibly be explained by the combination of this invention imparting better bone health, better gut health, better liver and better ovarian health. However, it is highly likely that estrogen is the hormone interlinking all these organs to successfully sustain prolonged hatching egg production. In essence, less incidence of degenerated and tumor-like morphology is likely resulted in a more sustained estrogen production by the hen and therefore longer egg-laying life.

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLES Example 1

Materials and Methods

A total of thirty 45-week-old broiler breeder hens (ROSS 308) were obtained from a commercial flock for the study. A basal broiler breeder laying diet was formulated as shown in Table 1. The calculated nutrient composition is shown in Table 2.

TABLE 1 Ingredient composition of the basal broiler breeder laying diets. Composition %, w/w Corn 66.9 Soybean meal 22.2 Oil fat 1.67 Ca Carbonate (ground 6.36 oyster shell) Dicalcium phosphate 1.8 Choline-Cl (70%) 0.1 Mineral Premix¹ 0.1 Copper sulfate 0.05 Vitamin Premix² 0.1 ¹Mineral premix provided (per kg of diet for treatment groups 1, 2 and 3): Cu 18 mg; I 1.1 mg; Fe 80 mg; Mn 150 mg; Zn 125 mg; and Se 0.25 mg. ²Refer to Table 2 for further detail.

TABLE 2 Vitamin premix composition (provided per kg of diet) Treatments 1 and 2 Treatment 3 1 = restricted feeding 3 = ad libitum feeding + Vitamin 2 = ad libitum feeding 25-OH-D3 + antioxidants A (IU) 10000 12000 D3 (IU) 2500 3000 E (mg) 100 150 K3 (mg) 3 5 B1 (mg) 3 5 B2 (mg) 8 14 B6 (mg) 6 8 B12 (mg) 0.03 0.03 Niacin (mg) 60 120 Pantothenic acid (mg) 18 30 Folic acid (mg) 1 4 Biotin (mg) 0.2 0.4 C (ascorbic acid) (mg) 0 150 25-OH-D3 (mcg) 0 69 Canthaxanthin (mg) 0 6

TABLE 3 Calculated nutrient composition (%) of the basal broiler breeder laying diets. Composition % w/w Crude protein 16 Crude fat 4.2 Calcium 3.1 Sodium 0.16 Total Phosphorus 0.64 Total ME 2910 kcal/kg

Diet was supplemented with or without 25-OH D3 at 69 mcg/kg diet in combination with antioxidants (ascorbic acid, canthaxanthin) and enriched levels of selected vitamins. Hens were randomly allocated to 3 treatment groups according to feeding regimen (restricted and ad libitum) as follows:

1. Basal diet—restricted feeding (140 g/day)

2. Basal diet—ad libitum feeding

3. Basal diet—ad libitum feeding+25-OH-D3 at 69 mcg/kg diet+antioxidants

They were individually-housed in wire cages placed in a controlled room with 14 h:10 h light:dark period and at a temperature of 25±3° C. Water was available ad libitum. The experimental period was lasted for 10 weeks. Three weeks after the feeding trial, some birds were used for relevant plasma parameter analyses. At the end of experiment, hens were euthanized and sacrificed for tissue sample collection for further studies:

A. 25-Hydroxy D3 and Antioxidants Lowered Mortality and Improved Egg Production, Ovarian Morphology and Plasma 17β Estradiol Level

Secretion of estradiol is the hallmark of successful ovulatory follicles. In addition to its role in triggering the preovulatory surge of gonadotropins, estradiol is an important intra-ovarian growth, differentiation, and survival factor. Inclusion of 25-hydroxy D3 and antioxidants reduced mortality and incidence of ovarian degeneration and ovarian-tumor-like morphology, increased egg production and sustained plasma estradiol levels in birds under ad libitum feed intake (Table 4).

TABLE 4 25-hydroxy D3 and antioxidants on mortality, egg production and plasma 17β-estradiol level of ad libitum-fed broiler breeder hens Ad libitum Ad feeding + Restricted libitum 25-OH-D3 + Pooled feeding feeding antioxidants SEM¹ Mortality (death/total) 1/7 4/10 2/10 Egg production (%) 36.8^(a) 17.6^(b) 28.5^(a) 6.72 Plasma 17β-estradiol 0.78^(a) 0.34^(c) 0.61^(b) 0.07 (nmole/mL) ^(a-c)Within a row, means without a common superscript differ (P < 0.05). ¹Pooled standard error of the mean.

Conclusions

Supplemental 25-hydroxy D3 and antioxidants ameliorated deleterious effects associated with overfeeding of broiler breeder females by:

a. lowering mortality rate and improving ovary function and therefore reproductive performance of overfed broiler breeder hens. 

1. A combination comprising: 25-Hydroxy vitamin D, ascorbic acid, Vitamin E and canthaxanthin for use in maintaining, increasing, or ameliorating the decrease of 17 β-estradiol levels in poultry, and/or increasing the productive egg laying life in poultry.
 2. A combination according to claim 1 further comprising at least one bio-active ingredient selected from the group consisting of Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, and Selenium.
 3. A feed or premix comprising a combination according to claim
 1. 4. A feed or premix according to claim 1 where the feed is for ad libitum feeding.
 5. A method of maintaining, increasing or ameliorating a decrease of 17 B-estradiol levels in poultry comprising providing a feed for the poultry comprising 25-Hydroxy vitamin D, ascorbic acid, Vitamin E and canthaxanthin.
 6. A method according to claim 5, further comprising at least one bio-active ingredient selected from the group consisting of Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, and Selenium.
 7. A method according to claim 4 wherein the feed is provided ad libidum.
 8. A method prolonging the productive egg laying life in poultry comprising administering a feed to the poultry comprising 25-Hydroxy vitamin D, ascorbic acid, Vitamin E and canthaxanthin.
 9. A method according to claim 8, further comprising at least one bio-active ingredient selected from the group consisting of Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, and Selenium.
 10. A method according to claim 8 wherein the feed is administered ad libidum. 